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United States Patent Application 20180105844
Kind Code A1
JEONG; Ki Jun ;   et al. April 19, 2018

METHOD OF PREPARING CINNAMALDEHYDE

Abstract

Provided is a method of preparing cinnamaldehyde by using a recombinant microorganism.


Inventors: JEONG; Ki Jun; (Daejeon, KR) ; KIM; Sun Chang; (Daejeon, KR) ; BANG; Hyun Bae; (Daejeon, KR) ; LEE; Yoon Hyeok; (Daejeon, KR) ; JUNG; Suk Chae; (Daejeon, KR)
Applicant:
Name City State Country Type

INTELLIGENT SYNTHETIC BIOLOGY CENTER

Daejeon

KR
Family ID: 1000003080728
Appl. No.: 15/511634
Filed: May 13, 2016
PCT Filed: May 13, 2016
PCT NO: PCT/KR2016/005085
371 Date: March 15, 2017


Current U.S. Class: 1/1
Current CPC Class: C12P 7/24 20130101; C12N 15/70 20130101; C12N 9/88 20130101; C12Y 403/01024 20130101; C12N 9/93 20130101; C12Y 602/01012 20130101; C12N 9/0008 20130101; C12Y 102/01044 20130101
International Class: C12P 7/24 20060101 C12P007/24; C12N 15/70 20060101 C12N015/70; C12N 9/88 20060101 C12N009/88; C12N 9/00 20060101 C12N009/00; C12N 9/02 20060101 C12N009/02

Foreign Application Data

DateCodeApplication Number
May 14, 2015KR10-2015-0067557

Claims



1. An expression cassette for producing cinnamaldehyde, the expression cassette comprising a pal (phenylalanine ammonia lyase) gene derived from Streptomyces maritimus, a 4 cl (4-coumarate:CoA ligase) gene derived from Streptomyces coelicolor, and a ccr (cinnamoyl Co-A reductase) gene derived from Arabidopsis thaliana.

2. The expression cassette of claim 1, wherein the pal gene has a nucleotide sequence represented by SEQ ID NO: 1or SEQ ID NO: 19.

3. The expression cassette of claim 1, wherein the pal gene encodes a protein having an amino acid sequence represented by SEQ ID NO: 2.

4. The expression cassette of claim 1, wherein the 4 cl gene has a nucleotide sequence represented by SEQ ID NO: 3.

5. The expression cassette of claim 1, wherein the 4 cl gene encodes a protein having an amino acid sequence represented by SEQ ID NO: 4.

6. The expression cassette of claim 1, wherein the car gene has a nucleotide sequence represented by SEQ ID NO: 5.

7. The expression cassette of claim 1, wherein the ccr gene encodes a protein having an amino acid sequence represented by SEQ ID NO: 6.

8. A vector for producing cinnamaldehyde, the vector comprising the expression cassette of any one of claims 1 to 7.

9. A transformant for producing cinnamaldehyde, the transformant comprising the vector of claim 8.

10. The transformant of claim 9, wherein the transformant is a microorganism belonging to Escherichia sp.

11. A method of preparing cinnamaldehyde, the method comprising culturing the transformant of claim 9.

12. The method of preparing cinnamaldehyde of claim 11, wherein the transformant is cultured in a medium comprising phenylalanine.

13. The method of preparing cinnamaldehyde of claim 11, wherein the transformant is cultured in a medium comprising casamino acid.
Description



BACKGROUND OF THE INVENTION

1. Field of the Invention

[0001] The present invention relates to a method of preparing cinnamaldehyde by using a recombinant microorganism.

2. Description of the Related Art

[0002] Cinnamaldehyde is the primary compound responsible for cinnamon flavor and odor, and accounts for 90% of cinnamon essential oil. Cinnamaldehyde is mainly prepared by adding a concentrated sodium bisulfite solution to cinnamon oil or cassia oil, separating a produced additive and washing it with ethanol, degrading a resulting product in dilute sulfuric acid or sodium carbonate aqueous solution, followed by steam distillation and vacuum distillation. Alternatively, cinnamaldehyde is prepared by adding acetaldehyde dropwise to a mixture of ben zaldehyde, water, and sodium hydroxide under stirring, extracting a reaction product with benzene, followed by fractional distillation under reduced pressure.

[0003] Cinnamaldehyde is used in a variety of applications. For example, Korean Patent Publication No. 2003-0033282describes antioxidant activity of cinnamaldehyde, and Korean Patent No. 10-0683113 describes a therapeutic effect of cinnamaldehyde on obesity. Korean Patent Publication No. 2013-0038000 describes a therapeutic effect of trans-cinnamaldehyde separated from cinnamon on hepatitis B.

[0004] It is necessary to study a method of effectively producing cinnamaldehyde which is known to have such various useful effects. Korean Patent No. 10-0624236 describes a method of preparing a cinnamaldehyde derivative by heating and refluxing a benzaldehyde derivative and vinyl acetate in an acetonitrile solvent in the presence of potassium carbonate and water.

[0005] Under this background, the present inventors have made many efforts to develop a method of efficiently producing cinnamaldehyde. As a result, they found that cinnamaldehyde may be prepared in a high yield by using a recombinant strain including a pal gene derived from Streptomyces maritimus, a 4 cl gene derived from Streptomyces coelicolor, and a ccr gene derived from Arabidopsis thallana, thereby completing the present invention.

SUMMARY OF THE INVENTION

[0006] An object of the present invention is to provide an expression cassette for producing cinnamaldehyde, including a pal (phenylalanine ammonia lyase) gene derived from Streptomyces maritimus, a 4 cl (4-coumarate:CoA ligase) gene derived from Streptomyces coelicolor, and a ccr (cinnamoyl Co-A reductase) gene derived from Arabidcpsis thallana.

[0007] Another object of the present invention is to provide a vector for producing cinnamaldehyde, including the expression cassette.

[0008] Still another object of the present invention is to provide a transformant for producing cinnamaldehyde, including the vector.

[0009] Still another object of the present invention is to provide a method of preparing cinnamaldehyde, including culturing the strain.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1 is a diagram showing a cinnamaldehyde production system according to an exemplary embodiment of the present invention.

[0011] FIG. 2 shows respective plasmids which were constructed for expression of PAL, 4 CL, and CCR used in an exemplary embodiment of the present invention.

[0012] FIG. 3 is SDS-PAGE showing quantities of purified PAL, 4 CL, and CCR proteins.

[0013] FIGS. 4 and 5 show concentrations of final products, cinnamate (CA) and cinnamaldehyde (CAD), respectively.

[0014] FIG. 6 shows optical density (OD.sub.600; open circle) measured at each time point and a concentration of cinnamaldehyde (CAB con.; closed circle), after a transformant was cultured in complex medium 1.

[0015] FIG. 7 snows optical density (OD.sub.600; open circle) measured at each time point and a concentration of cinnamaldehyde (CAE) con.; closed circle), after the transformant was cultured in complex medium 2.

[0016] FIG. 8 shows optical density (OD.sub.600; open circle) measured at each time point and a concentration of cinnamaldehyde (CAD con.; closed circle), after the transformant was cultured in defined medium 1.

[0017] FIG. 9 shows optrcal density (OD.sub.600; open circle) measured at each time point and a concentration of cinnamaldehyde; (CAD con.; closed circle), after the transformant was cultured in defined medium 2.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] In an aspect to achieve the above objects, the present invention provides an expression cassette for producing cinnamaldehyde including cinnamaldehyde biosynthesis genes, a vector including the same, a transformant including the same, or a method of preparing cinnamaldehyde by using the same.

[0019] The present inventors prepared a microorganism introduced with cinnamaldehyde biosynthesis genes, a pal (phenylalanine ammonia lyase) gene, a 4 cl (4-coumarate: CoA ligase) gene, and a ccr (cirmamoyl Co-A reductase) gene, and as a result, they found that cinnamaldehyde may be produced from the microorganism in a yield of 1.98 mg/L (FIGS. 6 to 9).

[0020] Further, they found that a combination of PAL derived from Streptomyces maritimus, 4 CL derived from Streptomyces coelicolor, and CCR derived from Arabidopsis thaliana shows a superior activity, compared to genes derived from other microorganisms (FIGS. 4 and 5).

[0021] Hereinafter, an expression cassette for producing cinnamaldehyde of the present invention will be described in detail.

[0022] The "cinnamaldehyde biosynthesis genes", as used herein, refer to a pal (phenylalanine ammonia lyase) gene, a 4 cl (4-coumarate: CoA ligase) gene, and a ccr (cinnamoyl Co-A reductase) gene.

[0023] The pal gene may be derived from Streptomyces maritimus. In an embodiment of the present invention, it was confirmed that PAL enzyme derived from Streptomyces maritimus has a superior enzymatic activity to PAL enzyme derived from Arabidopsis thaliana (FIG. 4).

[0024] For non-limiting example, the pal gene may include a nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 19. The pal gene may be represented by a sequence having 70% or higher, preferably 80% or higher, more preferably 90% or higher, or much more preferably 95% sequence homology to the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 19. It is apparent that the pal gene may also include a sequence modified by substitution, deletion, insertion, or a combination thereof one or more nucleotides in the above sequence.

[0025] For non-limiting example, the pal gene may encode a PAL protein having an amino acid sequence represented by SEQ ID NO: 2. The PAL protein may include all peptides, in which various amino acid sequences are added to N- or C-terminus of the amino acid sequence of SEQ ID NO: 2, as long as the protein has phenylalanine ammonia lyase activity. In addition, the PAL protein means a peptide further including a targeting sequence, tag, labeled residue, and an amino acid sequence designed for the specific purpose of increasing a half-life or stability of the peptide at the N- or C-terminus of the peptide represented by SEQ ID NO: 2. Furthermore, the PAL protein means a protein variant, in which part of amino acids in the amino acid sequence of SEQ ID NO: 2 is modified by a method such as addition, substitution, deletion, etc.

[0026] The 4 cl gene may be derived from Streptomyces coelicolor.

[0027] For non-limiting example, the 4 cl gene may include a nucleotide sequence represented by SEQ ID NO: 3, The 4 cl gene may be represented by a sequence having 70% or higher, preferably 80% or higher, more preferably 90% or higher, or much more preferably 95% sequence homology to the nucleotide sequence represented by SEQ ID NO: 3. It is apparent that the 4 cl gene may also include a sequence modified by substitution, deletion, insertion, or a combination thereof one or more nucleotides in the above sequence.

[0028] For non-limiting example, the del gene may encode a 4 CL protein having an amino acid sequence represented by SEQ ID NO: 4. The 4CL protein may include all peptides, in which various amino acid sequences are added to N- or C-terminus of the amino acid sequence of SEQ ID NO: 4, as long as the protein has 4-coumarate:CoA ligase activity. In addition, the 4 CL protein means a peptide further including a targeting sequence, tag, labeled residue, and an amino acid sequence designed for the specific purpose of increasing a half-life or stability of the peptide at the N- or C-terminus of the peptide represented by SEQ ID NO: 4. Furthermore, the 4 CL protein means a protein variant, in which part of amino acids in the amino acid sequence of SEQ ID NO: 4 is modified by a method such as addition, substitution, deletion, etc.

[0029] The ccr gene may be derived from Arabidopsis thaliana.

[0030] For non-limiting example, the ccr gene may include a nucleotide sequence represented by SEQ ID NO: 5. The ccr gene may be represented by a sequence having 70% or higher, preferably 80% or higher, more preferably 90% or higher, or much more preferably 95% sequence homology to the nucleotide sequence represented by SEQ ID NO: 5. It is apparent that the ccr gene may also include a sequence modified by substitution, deletion, insertion, or a combination thereof one or more nucleotides in the above sequence.

[0031] For non-limiting example, the ccr gene may encode a CCR protein having an amino acid sequence represented by SEQ ID NO: 6. The CCR protein may include all peptides, in which various amino acid sequences are added to N- or C-terminus of the amino acid sequence of SEQ ID NO: 6, as long as the protein has cinnamoyl Co-A reductase activity. In addition, the CCR protein means a peptide further including a targeting sequence, tag, labeled residue, and an amino acid sequence designed for the specific purpose of increasing a half-life or stability of the peptide at the N- or C-terminus of the peptide represented by SEQ ID NO: 6. Furthermore, the CCR protein means a protein variant, in which part of amino acids in the amino acid sequence of SEQ ID NO: 6 is modified by a method such as addition, substitution, deletion, etc.

[0032] The polynucleotide may be modified by substitution, deletion, insertion, or a combination thereof one or more nucleotides. When the nucleotide sequence is prepared by chemical synthesis, a synthesis method widely known in the art, for example, a method described in a literature (Engels and Uhlmann, Angew Chem IntEd Engl., 37: 73-127, 1988) may be used. Triester, phosphite, phosphoramidite and H-phosphate methods, PCR and other autoprimer methods, oligonucleotide synthesis methods on solid supports may be used.

[0033] In an embodiment of the present invention, when 4 CL derived from Streptornyces coelicolor and CCR derived from Arabidcpsis thaliana are used in combination, superior enzymatic activity was obtained, compared to a combination of 4 CL derived from Arabidopsis thaliana and CCR derived from Arabidopsis thaliana (FIG. 5)

[0034] The term "homology", as used herein in relation to the sequence, refers to a degree of matching with a given amino acid sequence or nucleotide sequence, and the homology may be expressed as a percentage. In the present invention, a homology sequence having an activity which is identical or similar to the given amino acid sequence or nucleotide sequence is expressed as "% homology". The homology sequence may be determined by, for example, a standard software, specifically, BLAST 2.0, which calculates the parameters such as score, identity, similarity, etc., or by comparing the sequences in a Southern hybridization experiment under defined stringent conditions, and defining appropriate hybridization conditions are within the skill of the art (Sambrook et al. , 1989, infra.), and may be determined by a method well known to those skilled in the art.

[0035] The gene may be codon-optimized with respect to the transformant. The codon optimization refers to replacement of codons of the genes by codons of high frequency in a host for efficient expression of the genes in the host. As long as a method for optimization is used to increase protein expression in the transformant, any method known in the art may be used without limitation.

[0036] The term "expression cassette", as used herein, refers to a unit cassette capable of expressing cinnamaldehyde, because of including the cinnamaldehyde biosynthesis genes. In the present invention, the expression cassette may be interchangeable with an expression construct. With respect to the objects of the present invention, the expression cassette according to the present invention may be introduced into a strain to produce cinnamaldehyde.

[0037] In another aspect, the present invention provides a vector for producing cinnamaldehyde, including the expression cassette.

[0038] The term "vector" or "expression vector", as used herein, refers to a DNA construct including a nucleotide sequence of a polynucleotide encoding a target protein, which is operationally linked to a suitable regulatory sequence so that the target protein may be expressed in an appropriate host. The regulatory sequence includes a promoter to direct transcription, an arbitrary operator sequence to regulate such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence to regulate termination of transcription and translation. The vector, after being transformed into an appropriate host cell, may be replicated or function irrespective of the host genome, or may be integrated into the genome itself.

[0039] The vector may include the cinnamaldehyde biosynthesis genes operably linked thereto. The term "operably linked", as used herein, refers to a functional linkage between a nucleotide expression control sequence and a nucleotide sequence encoding a target protein or RNA in such a manner as to allow general functions. For instance, a promoter and a nucleotide sequence encoding a protein or RNA are operably linked to each other to affect expression of a coding sequence. The operable linkage to a recombinant vector may be prepared using a genetic recombinant technique well known in the art, and site-specific DNA cleavage and ligation may be carried out using enzymes that are generally known in the art.

[0040] The expression vector used in the present invention is not particularly limited, as long as it may replicate in a host cell, and the expression vector may be any vector known in the art. Example of the vector commonly used may be a natural or recombinant plasmid, cosmid, virus and bacteriophage. For example, as the phage vector or the cosmid vector, pWE15, .lamda.M13, .lamda.MBL3, .lamda.MBL4, .lamda.IXII, .lamda.ASHII, .lamda.APII, .lamda.t10, .lamda.t11, Charon4A, and Charon21A, etc. may be used. As the plasmid vector, pET type, pTrc type, pBR type, pUC type, pBluescr iptII type, pGEM type, pTZ type, pCL type, pMAL type, pHT type, etc. may be used. The vector usable in the present invention is not particularly limited, and any known expression vector may be used. Specifically, pET22b, pTrc99a, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, pMAL-p2x, or pHT43 vector, etc. may be used.

[0041] The vector of the present invention may be integrated into the chromosome by homologous recombination, and therefore, it may further include a selection marker to ensure the chromosomal insertion.

[0042] The appropriate expression vector may include a signal sequence or a leader sequence for targeting membranes or secretion as well as expression regulatory elements, such as a promoter, an operator, an initiation ccdon, a stop codon, a polyadenylation signal and an enhancer, and may be constructed in various forms depending on the purpose thereof. The initiation and stop codons are generally considered as part of a nucleotide sequence encoding a target protein, and are necessary to be functional in an individual to whom a genetic construct has been administered, and must be in frame with the coding sequence. The promoter of the vector may be constitutive or inducible. Further, the expression vector may include a selection marker that allows the selection of host cells including the vector, and a replicable expression vector may include a replication origin. The vector may be self-replicable, or may be integrated into the host DNA.

[0043] Furthermore, the expression vector may further include a protein tag removed by using endopeptidase for easy detection of cinnamaldehyde. The tag refers to a molecule which exhibits a quantifiable activity or characteristic, and examples thereof may include fluorescent molecules including a chemical fluorescence such as fluorescein, and a polypeptrde fluorescence such as green fluorescent protein (GFP) and related proteins; and an epitope tag such as a Myc tag, a Flag tag, a histidine tag, a leucine tag, an IgG tag, a streptavidin tag, etc. When the epitope tag is used, a peptide tag consisting of preferably 6 or more amino acid residues, and more preferably 8 to 50 amino acid residues may be used.

[0044] A method of preparing the vector is not particularly limited, and any method commonly used in the art may be used.

[0045] In still another aspect, the present invention provides a transformant for producing cinnamaldehyde, including the vector.

[0046] The "producing", as used herein, is intended to include secretion of cinnamaldehyde out of cells, for example, secretron of cinnamaldehyde into a medium, as well as production of cinnamaldehyde within a strain.

[0047] The "transformation", as used herein, refers to introduction of a vector including a polynucleotide encoding a target protein into a host cell in such a way that the protein encoded by the polynucleotide may be expressed in the host cell.

[0048] As long as the transformation method is a known method in the art, the method may be used without limitation. As long as the transformation method is able to produce cinnamaldehyde, the method may include, but is not particularly limited to, CaCl.sub.2 precipitation, a Hanahan method that is an improved CaCl.sub.2 method by using DMSO (dimethyl sulfoxide) as a reducing material, electroporation, calcium phosphate precipitation, protoplast fusion, agitation using silicon carbide fiber, Agrobacterium-mediated transformation, PEG-mediated transformation, dextran sulfate, lipofectamine, Land desiccation/inhibition-mediated transformation.

[0049] As long as the transformed polynucleotide may be expressed in the host cell, it may be either integrated into or placed in the chromosome of the host cell, or exist extrachromosomally. Further, the polynucleotide includes DNA and RNA encoding the target protein. The polynucleotide may be introduced in any form, as long as it may be introduced into the host cell and expressed therein. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a gene construct including all elements required for its autonomous expression.

[0050] A microorganism which may be used as the transformant may be a microorganism belonging to Escherichia sp., Shigella sp., Citrobacter sp., Salmonella sp., Enterobacter sp. Yersinia sp., Klebsiella sp., Erwinia sp., Corynebacterium sp., Brevibacterium sp., Lactobacillus sp., Selenomanas sp., Vibrio sp., Pseudomonas sp., Streptomyces sp., Arcanobacterium sp., Alcaligenes sp, etc., but is not limited thereto. Specifically, the microorganism may be a microorganism belonging to Escherichia sp.

[0051] In still another aspect, the present invention provides a method of preparing cinnamaldehyde, including culturing the transformant.

[0052] In a specific embodiment of the present invention, the transformant having cinnamaldehyde productivity was cultured and then, a production amount of cinnamaldehyde included in a culture was measured. As a result, it was confirmed that cinnamaldehyde may be produced from the microorganism in a yield of 1.98 mg/L (FIGS. 6 to 8).

[0053] The term "culturing", as used herein, refers to a method of growing the microorganism under artificially controlled environmental conditions. In the present invention, the method of culturing the transformant may be performed by using any method widely known in the art. Specifically, as long as the culturing may be performed to express and produce cinnamaldehyde of the present invention, it may be performed by, but is not particularly limited to, a known batch culture, continuous culture, fed-batch culture, etc.

[0054] In this regard, the culture conditions are not particularly limited, but an optimal pH (e.g., pH 5 to pH 9, preferably pH 6 to pH 3, and. most, preferably pH 6.8) may be maintained by using a basic chemical (e.g., sodium hydroxide, potassium hydroxide, or ammonia) or an acidic chemical (e.g., phosphoric acid or sulfuric acid). An aerobic condition may be maintained by adding oxygen or oxygen-containing gas mixture to the culture. The culture temperature may be maintained at 20.degree. C. to 45.degree. C., and preferably at 25.degree. C. to 40.degree. C., and the culturing may be preferably performed for about 10 hrs to 160 hrs.

[0055] Media used for the culturing need to meet requirements for growth of particular strains in an appropriate manner by controlling temperature, pH. etc. in a common medium containing appropriate carbon sources, nitrogen sources, amino acids, vitamin, etc.

[0056] A carbon source to be used may be sugar and carbohydrate (e.g., glucose, sucrose, lactose, fructose, maltose, molasses, starch, and cellulose), oil and fat (e.g., soybean oil, sunflower oil, peanut oil, and coconut oil), a fatty acid (e.g., palmitic acid, stearic acid, and linolenic acid), an alcohol (e.g., glycerol and ethanol), and an organic acid (e.g., acetic acid). The carbon sources may be used alone or in a mixture.

[0057] A nitrogen source may be a nitrogen-containing organic compound (e.g., peptone, yeast extract, meat extract, malt extract, corn steep liquor, soy meal, and urea) or an inorganic compound (e.g., ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium, nitrate). The nitrogen source may be used alone or in a mixture.

[0058] A phosphorus source may be potassium dihydrogen phosphate, dipotassium hydrogen phosphate, corresponding sodium-containing salts, etc. The phosphorus source may be used, alone or in a mixture. The culture medium may include essential growth-promoting materials such as metal salts (e.g., magnesium sulfate or iron sulfate), amino acids, and vitamins.

[0059] Further, proper precursors may be added to the culture medium. These materials may be added to the culture during culturing by an appropriate method in a batch, fed-batch, or continuous mode, but are not limited thereto. pH of the culture may be adjusted by a proper method using a basic compound such as sodium hydroxide, potassium hydroxide, or ammonia, or an acidic compound such as phosphoric acid or sulfuric acid.

[0060] in the present invention, the medium may be used in the same meaning as the culture medium.

[0061] For example, the preparation method may further include collecting cinnamaldehyde from the cultured transformant or a culture thereof.

[0062] The term "culture", as used herein, refers to a resulting material obtained by culturing the microorganism., and may include all of the medium, the cultured microorganism, and materials secreted by the cultured microorganism. For example, the culture may include nutrient sources required to culture the strain, for example, inorganic salts, amino acids, vitamins, nucleic acids and/or other general components included in a culture medium (or culture liquid), in addition to carbon sources, nitrogen sources, etc. Further, the culture may include, for example, enzymes produced/secreted by the strain, etc.

[0063] Since cinnamaldehyde produced by the culturing may be secreted into the medium or may remain within cells, the culture may include cinnamaldehyde produced by culturing the microorganism.

[0064] A method of collecting cinnamaldehyde produced in the culturing of the present invention may be performed to collect cinnamaldehyde from the culture liquid by using an appropriate method known in the art according to the culturing method, for example, batch, continuous or fed-batch culture. Specifically, the collecting method may be, for example, centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (e.g., ammonium sulfate precipitation), chromatography (e.g., ion exchange, affinity, hydrophobic, and size exclusion chromatography), etc.

[0065] For example, the preparation method may further include purifying the collected cinnamaldehyde. The purifying may be performed by general chromatography including immunoaffinity chromatography, receptor affinity chromatography, hydrophobic interaction chromatography, lectin affinity chromatography, size exclusion chromatography, cation or anion exchange chromatography, high performance liquid chromatography (HPLC), reverse phase HPLC, etc. Further, the desired protein, which is the fusion protein with a specific tag, label, or chelate moiety, may be purified after being recognized by specific binding partners or drug. The purified protein may be cut as desired part of protein, or it may remain as it is. The desired form of the protein having additional amino acid may be produced from, the cutting process by cutting the fusion protein.

[0066] For example, the transformant may be cultured in a medium containing phenylalanine. The pal gene included in the transformant utilizes phenylalanine as a substrate. Therefore, when the transformant is cultured in the medium containing phenylalanine, cinnamaldehyde may be produced with a superior efficiency.

[0067] For example, the transformant may be cultured in a medium containing casamino acid. In an embodiment of the present invention, when the transformant including cinnamaldehyde biosynthesis genes was cultured in the medium containing casamino acid, a maximum production yield was as high as 1.98 mg/L. In contrast, when the transformant including cinnamaldehyde biosynthesis genes was cultured in a medium containing no casamino acid, a maximum production yield was 1.23 mg/L.

[0068] Hereinafter, the present invention will be described in detail with reference to the following Examples. However, these Examples are for illustrative purposes only, and the invention is not intended to be limited by these Examples.

EXAMPLE 1

Preparation of Strain and Plasmid for Recombinant Gene Expression

[0069] First, from Arabidopsis thaliana cDNA, pal (phenylalanine ammonia-lyase, SEQ ID NO: 17), 4 cl (4-coumarate:CoA ligase, SEQ ID NO: 18), and ccr (cinnamoyl-CoA reductase, SEQ ID NO: 5) genes were obtained by polymerase chain reaction using BHB 22-F and BHB 22-R, BHB 20-F and. BHB 20-R, BHB 19-F and BHB 19-R of sequences described in the following Table 1.

[0070] Further, from Streptomyces maritimus pal gene synthesized by codon optimization, pal gene (SEQ ID NO: 19) was obtained by polymerase chain reaction using BHB 31-F and BHB 3I-R of sequences described in the following Table 1. Further, from Streptomyces coelicolor genomic DNA, 4 cl gene (SEQ ID NO: 3) was (obtained by polymerase chain reaction using BHB 21-F and BHB 21-R of sequences described in the following Table 1.

TABLE-US-00001 TABLE 1 BHB 19-F GCATCTAGAAACACAAACAAGGAAGGAAGATAAATGCACCACCACCAC SEQ ID NO: 7 CACCACCACCACATGCCAGTCGACGTAGCC BHB 19-R ATGCGCGGCCGCTTATCAAGACCCGATCTTAATGCCATTTTC SEQ ID NO: 8 BHB 20-F GCATCTAGACCGAAATCAAAAGGAACACCAACGTATGCACCACCACCA SEQ ID NO: 9 CCACCACCACCACATGGCGCCACAAGAACAAG BHB 20-R ATGCGCGGCCGCTTATCACAATCCATTTGCTAGTTTTGCCC SEQ ID NO: 10 BHB 21-F GCATCTAGACGAATACCTGGAGGACCTAAACAGTATGCACCACCACCA SEQ ID NO: 11 CCACCACCACCACATGTTCCGCAGCGAGTAC BHB 21-R ATGCGCGGCCGCTTATCATCGCGGCTCCCTGAGCT SEQ ID NO: 12 BHB 22-F GCATCTAGACACCTTAAGGAGGTCTATCTTTCATATGCACCACCACCA SEQ ID NO: 13 CCACCACCACCACATGGAGATTAACGGGGCAC BHB 22-R ATGCGCGGCCGCTTATCAACATATTGGAATGGGAGCTCC SEQ ID NO: 14 BHB 31-F GCATTCTAGACCCAACGAAGGGGGAACCACACAATATGCACCACCACC SEQ ID NO: 15 ACCACCACCACCACACCTTCGTTATTGAACTGGATATGAATGTTACCC BHB 31-R ATGCGCGGCCGCTTATCAGTGTGCTGCCACGGCTG SEQ ID NO: 16

[0071] Respective PCR products were digested with restriction enzymes, XbaI and HindIII, and each of the products was ligated to a vector described in the following Table 2 and FIG. 2

TABLE-US-00002 TABLE 2 Relevant Characteristics Strain E. coli MG1655 F.sup.- .lamda..sup.- ilvG.sup.-rfb.sup.-50rph.sup.-1 E. coli BL21(DE3) F ompT gal dcm lon hsdS.sub.B(r.sub.B.sup.- m.sub.B.sup.-) .lamda.(DE3 [lacI lacUV5- T7 gene 1 ind1 sam7 nin5]) E. coli W3110 F.sup.- .lamda..sup.- rph.sup.-1 INV(rrnD, rrnE) E. coli NST74 E. coli W3110 derivative (aroF aroG tyrR pheA pheAo) Plasmid pET22b Amp.sup.R, T7 promoter pTrc99a Amp.sup.R, trc promoter pHB-I01 pET22b derivative, His.sub.8-tagA. thaliana PAL pHB-I02 pET22b derivative, His.sub.8-tagA. thaliana 4CL pHB-I03 pET22b derivative, His.sub.8-tagA. thaliana CCR pHB-I04 pET22b derivative, His.sub.8-tagS. maritimus PAL pHB-I05 pET22b derivative, His.sub.8-tagS. coelicolor 4CL pHB-P01 pTrc99a derivative, FLAG-tag S. maritimus PAL, His.sub.8-tagS. coelicolor 4CL, and His.sub.8-tagA. thaliana CCR

[0072] After completion of ligation, pHB-I01, pHB-I02, pHB-I03, pHB-I04, or pHB-I05 vector was transformed into BL21(DE3) via E. coli strain MG1655. The pHB-P01 vector was transformed into E. coli strain NST74.

EXAMPLE 2

Isolation and Purification of PAL, 4CL, and CCR proteins

[0073] Each of E. coli strains, BL21(DE3) and NST74, was inoculated in an LB (Luria-Bertani) medium containing 2% glucose and 100 .mu.g/mL ampicillin. The strains were incubated under conditions of 37.degree. C. and 200 rpm for 12 hrs, and then each 1/100 volume thereof was transferred to a fresh LB medium. Thereafter, each of the strains was incubated under the same conditions, until OD.sub.600 reached 0.6.

[0074] Thereafter, BL21(DE3) strain was adapted under conditions of 25.degree. C. and 200 rpm for 30 min for protein production, and then 1 mM IPTG (isopropyl-.beta.-D-thiogalactopyranoside) was added thereto, followed by further incubation for 6 hrs. Meanwhile, NST74 strain was incubated under conditions of 37.degree. C. and 200 rpm after addition of 1 mM IPTG.

[0075] Thereafter, the culture medium was centrifuged under conditions of 4.degree. C. and 6000 rpm for 10 min, and then a supernatant was discarded. A pellet was resuspended in a buffer (50 mM potassium phosphate, 300 mM sodium chloride, pH 7.0), and disrupted using a sonicator to obtain a cell suspension. Thereafter, this cell suspension was centrifuged under conditions of 4.degree. C. and 10000 rpm for 10 min, and a water-soluble supernatant was obtained.

[0076] Thereafter, from the cell suspension, PAL, 4 CL, and CCR proteins having 8.times. histidine tag were purified by IMAC (immobilized metal affinity chromatography).

[0077] Further, the water-soluble supernatant was filtered using a 0.45 .mu.m filter, and added to Talon.RTM. metal affinity resin pretreated with a binding buffer (50 mM potassium phosphate, 300 mM sodium chloride, pH 7.0). Thereafter, the resin was washed with 10 mL of a washing buffer (50 mM potassium phosphate, 300 mM sodium chloride, 15 mM imidazole, pH 7.0), and then final proteins were purified by using 1 mM elation buffer (50 mM potassium phosphate, 300 mM sodium chioride, 150 mM imidazole, pK 7.0).

EXPERIMENTAL EXAMPLE 1

Quantification of PAL, 4CL, and CCR proteins

[0078] The purified proteins were quantified by densitometry using 12% (w/v) SDS-PAGE (sodium. dodecyl sulfate-polyacrylamide gel electrophoresis) and total protein assay using BSA (bovine serum albumin), and the results are shown in FIG. 3.

[0079] In FIG. 3, lane M represents the protein size. Lane 1 to lane 3 represent a total protein (total), a soluble protein (soluble), an eluted protein after purification (elution) of Arabidopsis thaliana PAL enzyme, respectively. Lane 4 to lane 6 represent a total protein (total), a soluble protein (soluble), an eluted protein after purification (elution) of Arabidopsis thaliana 4CL enzyme, respectively. Lane 7 to lane 9 represent a total protein (total), a soluble protein (soluble), an eluted protein after purification (elution) of Arabidopsis thaliana CCR enzyme, respectively. Lane 10 to lane 12 represent, a total protein (total), a soluble protein (soluble), an eluted protein after purification (elution) of Streptomyces maritimus PAL enzyme, respectively. Lane 13 to lane 15 represent a total protein (total), a soluble protein (soluble), an eluted protein after purification (elution) of Streptomyces coelicolor 4 CL enzyme, respectively.

[0080] With regard to the size of each protein, Arabidopsis thaliana PAL was 78 kDa (solid arrow), Arabidopsis thaliana 4 CL was 61 kDa (dashed arrow), Arabidopsis thaliana CCR was 37 kDa (closed triangle), Streptomyces maritimus PAL was 56 kDa (open triangle), Streptomyces coelicolor 4 CL was 55 kDa (open triangle), indicating that PAL, 4 CL, and CCR proteins were produced well.

EXPERIMENTAL EXAMPLE 2

Analysis of Enzymatic Activity

[0081] Activities of PAL, 4 CL, and CCR proteins purified in Example 2 were analyzed by extracellular reaction.

[0082] First, to analyze the activity of PAL, 100 mM Tris-HCl, 0.2 mM phenylalanine, and 200 .mu.g/mL of purified PAL enzyme were reacted. To analyze the activities of 4 CL and CCR, 400mM Tris-HCl, 5 mM ATP, 5 mM magnesium chloride, 0.3 mM Coenzyme A, 0.5 mM trans-cinnamate, and each 50 .mu.g/mL of the purified 4 CL and CCR enzymes were reacted.

[0083] After the reaction was allowed at 30.degree. C. for 1 hr, cinnamate produced by PAL enzyme and cinnamaldehyde produced by 4 CL and CCR enzymes were analyzed by reverse-phase high-performance liquid chromatography (reverse-phase HPLC). In detail, in a ZORBAX Eclipse AAA column (150.times.4.6 mm; 3.5 .mu.m; Agilent, Calif., USA), a mobile phase A was 0.1% trifluoroacetic acid, and a mobile phase B was acetonitrile. A ratio of acetonitrile was gradually increased from 10% for initial 1min to 70% for 9 min. A temperature of the column was 40.degree. C., and a flow rate was maintained at 1 mL/min.

[0084] Standard solutions (1, 10, 50, 100, and 200 mg/L of cinnamate, and 0.41, 4.1, 41, 82, and 136.7 mg/L of cinnamaldehyde) were used to create standard curves, which were used to determine the quantities of the final products, cinnamate (CA in FIG. 4) and cinnamaldehyde (CAD in FIG. 5), respectively.

[0085] As shown in FIG. 4, PAL derived from Streptomyces maritimus was found to have higher activity than PAL derived from Arabidopsis thaliana. Further, as shown in FIG. 5, when a combination of 4 CL derived from Streptomyces maritimus and CCR derived from Arabidopsis thaliana was used, a high enzymatic activity was observed, compared to a combination of 4 CL derived from Arabidopsis thaliana and CCR derived from Arabidopsis thaliana.

EXPERIMENTAL EXAMPLE 3

High-Cell Density Cultivation

[0086] First, 200 mL of pHB-PO1-including E. coli strain NST74 which was adapted to an R/2 semi-defined medium (6.75 g/L potassium dihydrogen phosphate, 2 g/L ammonium phosphate dibasic, 0.85 g/L citric acid, 0.7 g/L magnesium sulfate heptahydrate, 5 mL/L trace metal solution (TMS; 10 g/L iron sulfate heptahydrate, 2.2 g/L zinc sulfate heptahydrate, 2g/L calcium chloride dehydrate, 1 g/L copper sulfate pentahydrate, 0.58 g/L manganese sulfate pentabydrate, 0.1 g/L ammonium heptamolybdate tetrahydrate, 0.02 g/L sodium tetraborate decahydrate, pH 6.8) was incubated at 37.degree. C. and 200 rpm for 12 hrs.

[0087] Thereafter, the strain was inoculated in 1.8 L of the same medium at a total volume of 2 L, and high-density cultivation was performed in a 5 L-bioreactor. When pH was lower than 6.77, 50% (v/v) ammonia was introduced. When pH was higher than 6.86, a feeding solution was introduced. As the feeding solution, a complex feeding solution 1 (500 g/L glucose, 75 g/L yeast extract, 20 g/L magnesium sulfate heptahydrate), a complex feeding solution 2 (500 g/L glucose, 100 g/L casamino acid, 20 g/L magnesium sulfate heptahydrate), a defined feeding solution 1 (700 g/L glucose, 20 g/L magnesium sulfate heptahydrate), or a defined feeding solution 2 (500 g/L glucose, 20 g/L magnesium, sulfate heptahydrate, 0.8 1 g/L phenylalanine) was used.

[0088] The temperature was maintained, at 37.degree. C., and dissolved oxygen (DO) was maintained at 40% by supplying oxygen, after an agitation rate was increased to 1000 rpm. When optical density (OD.sub.600) reached 60, 1 mM IPTG was introduced to produce proteins (enzymes). An anti-foaming agent was introduced by manually after sterilization.

[0089] The cultured E. coli strain was separately collected at an optical density (OD.sub.600) of 4 at each time point, and used for protein analysis. A supernatant of the culture was filtered using a 0.22 .mu.m filter, and used in quantification of cinnamaldehyde by reverse-phase HPLC.

[0090] FIG. 6 shows optical density (OD.sub.600; open circle) measured at each time point and the concentration of cinnamaldehyde (CAD con.; closed circle), after complex feeding solution 1 was used as the feeding solution. A maximum production yield was 1.2 3 mg/L.

[0091] FIG. 7 shows optical density (OD.sub.600; open circle) measured at each time point and the concentration of cinnamaldehyde (CAD con.; closed circle), after complex feeding solution 2 was used as the feeding solution. A maximum production yield was 1.98 mg/L.

[0092] FIG. 8 shows optical density (OD.sub.600; open circle) measured at each time point and the concentration of cinnamaldehyde (CAD con.; closed circle), after defined feeding solution 1 was used as the feeding solution. A maximum production yield was 0.71 mg/L.

[0093] FIG. 9 shows optical density (OD.sub.600; open circle) measured at each time point and the concentration of cinnamaldehyde (CAD con.; closed circle), after defined feeding solution 2 was used as the feeding solution. A maximum production yield was 1.05 mg/L.

[0094] Based on the above description, it will be understood by those skilled in the art that the present invention may be implemented in a different specific form without changing the technical spirit or essential characteristics thereof. Therefore, it should be understood that the above embodiment is not limitative, but illustrative in all aspects. The scope of the invention is defined by the appended claims rather than by the description preceding them, and therefore all changes and modifications that fall within metes and bounds of the claims, or equivalents of such metes and bounds are therefore intended to be embraced by the claims.

[0095] Effect of the Invention

[0096] An expression cassette for producing cinnamaldehyde according to the present invention, a vector including the same, a transformant including the same, and a method of preparing cinnamaldehyde by using the same may be used to produce cinnamaldehyde with high efficiency.

Industrial Applicability

[0097] Accordingly, the present invention provides an expression cassette for producing cinnamaldehyde, a vector including the same, a transformant including the same, and a method of preparing cinnamaldehyde by using the same

Sequence CWU 1

1

1911569DNAStreptomyces maritimus 1atgaccttcg tcatagagct cgacatgaac gtcacgctcg accaacttga ggacgcggcg 60cgacagcgca cgcccgtgga gctgtccgca cccgtccgct cccgcgtccg cgcctcgcgc 120gacgtgttgg tgaagttcgt gcaggacgaa cgtgtcatct acggggtcaa caccagcatg 180gggggcttcg tcgaccacct cgtcccggtg tcccaggccc ggcagctcca ggagaacctg 240atcaacgcgg tcgccaccaa cgtgggggcg tatctggacg acacgaccgc ccggaccatc 300atgctgtccc gcatcgtgtc gctggcgcgc gggaactccg cgatcacccc ggcgaatctg 360gacaagctgg tggccgtact caacgccggg atcgtgccgt gcatcccgga gaagggctct 420ttgggcacca gcggtgacct cggcccgctg gccgcgatcg ccctggtgtg cgcggggcag 480tggaaggccc gctacaacgg tcagatcatg cccgggcggc aggccctgtc cgaggccggc 540gtcgagccga tggagctgag ctacaaggat ggcctggccc tgatcaacgg cacgtcaggc 600atggtcggcc tgggcaccat ggtcctccag gccgcgcgcc ggctcgtgga ccgctacctg 660caggtgtccg cgttgtcggt cgagggcctg gcaggcatga cgaaaccgtt cgaccctcgc 720gtgcacggcg tcaagccgca ccgcgggcag cgtcaggtgg cctcgcggtt gtgggagggg 780cttgccgact cgcacctggc ggtcaacgaa ctggacaccg agcagaccct ggccggagag 840atgggcacgg tcgccaaggc cggttcgctg gcgatcgagg acgcctactc catccggtgc 900acgccgcaga tcctcggtcc cgtggtcgat gtgctggacc ggatcggggc gaccctgcag 960gacgagctga actcctccaa cgacaacccg atcgtcctgc cggaggaggc ggaggtgttc 1020cacaacgggc acttccacgg ccagtacgtg gccatggcca tggaccacct gaacatggcc 1080ctggccaccg tgaccaatct cgccaaccgg cgcgtggacc gcttcctgga caagagcaac 1140agcaacgggc tgcccgcctt cctgtgccgg gaagatccgg gactgcgcct gggcctgatg 1200ggcggccagt tcatgaccgc gtcgatcacc gcggagaccc gcaccctgac cattccgatg 1260tcggtgcagt ccctcacgag tacggcggac ttccaggaca tcgtgtcctt cggattcgtc 1320gccgcccgcc gcgcccggga ggtactcacc aacgctgcct acgtggtggc cttcgagctg 1380ctgtgcgcct gccaggccgt cgacatccgc ggcgcggaca aactgtcctc cttcacccgc 1440ccgctctatg agcgcacccg caagatcgtg ccgttcttcg accgggacga gaccatcacc 1500gactacgtcg agaagctggc ggccgacctg atcgcgggcg agcccgtcga cgctgccgtg 1560gcggcgcac 15692523PRTStreptomyces maritimus 2Met Thr Phe Val Ile Glu Leu Asp Met Asn Val Thr Leu Asp Gln Leu 1 5 10 15 Glu Asp Ala Ala Arg Gln Arg Thr Pro Val Glu Leu Ser Ala Pro Val 20 25 30 Arg Ser Arg Val Arg Ala Ser Arg Asp Val Leu Val Lys Phe Val Gln 35 40 45 Asp Glu Arg Val Ile Tyr Gly Val Asn Thr Ser Met Gly Gly Phe Val 50 55 60 Asp His Leu Val Pro Val Ser Gln Ala Arg Gln Leu Gln Glu Asn Leu 65 70 75 80 Ile Asn Ala Val Ala Thr Asn Val Gly Ala Tyr Leu Asp Asp Thr Thr 85 90 95 Ala Arg Thr Ile Met Leu Ser Arg Ile Val Ser Leu Ala Arg Gly Asn 100 105 110 Ser Ala Ile Thr Pro Ala Asn Leu Asp Lys Leu Val Ala Val Leu Asn 115 120 125 Ala Gly Ile Val Pro Cys Ile Pro Glu Lys Gly Ser Leu Gly Thr Ser 130 135 140 Gly Asp Leu Gly Pro Leu Ala Ala Ile Ala Leu Val Cys Ala Gly Gln 145 150 155 160 Trp Lys Ala Arg Tyr Asn Gly Gln Ile Met Pro Gly Arg Gln Ala Leu 165 170 175 Ser Glu Ala Gly Val Glu Pro Met Glu Leu Ser Tyr Lys Asp Gly Leu 180 185 190 Ala Leu Ile Asn Gly Thr Ser Gly Met Val Gly Leu Gly Thr Met Val 195 200 205 Leu Gln Ala Ala Arg Arg Leu Val Asp Arg Tyr Leu Gln Val Ser Ala 210 215 220 Leu Ser Val Glu Gly Leu Ala Gly Met Thr Lys Pro Phe Asp Pro Arg 225 230 235 240 Val His Gly Val Lys Pro His Arg Gly Gln Arg Gln Val Ala Ser Arg 245 250 255 Leu Trp Glu Gly Leu Ala Asp Ser His Leu Ala Val Asn Glu Leu Asp 260 265 270 Thr Glu Gln Thr Leu Ala Gly Glu Met Gly Thr Val Ala Lys Ala Gly 275 280 285 Ser Leu Ala Ile Glu Asp Ala Tyr Ser Ile Arg Cys Thr Pro Gln Ile 290 295 300 Leu Gly Pro Val Val Asp Val Leu Asp Arg Ile Gly Ala Thr Leu Gln 305 310 315 320 Asp Glu Leu Asn Ser Ser Asn Asp Asn Pro Ile Val Leu Pro Glu Glu 325 330 335 Ala Glu Val Phe His Asn Gly His Phe His Gly Gln Tyr Val Ala Met 340 345 350 Ala Met Asp His Leu Asn Met Ala Leu Ala Thr Val Thr Asn Leu Ala 355 360 365 Asn Arg Arg Val Asp Arg Phe Leu Asp Lys Ser Asn Ser Asn Gly Leu 370 375 380 Pro Ala Phe Leu Cys Arg Glu Asp Pro Gly Leu Arg Leu Gly Leu Met 385 390 395 400 Gly Gly Gln Phe Met Thr Ala Ser Ile Thr Ala Glu Thr Arg Thr Leu 405 410 415 Thr Ile Pro Met Ser Val Gln Ser Leu Thr Ser Thr Ala Asp Phe Gln 420 425 430 Asp Ile Val Ser Phe Gly Phe Val Ala Ala Arg Arg Ala Arg Glu Val 435 440 445 Leu Thr Asn Ala Ala Tyr Val Val Ala Phe Glu Leu Leu Cys Ala Cys 450 455 460 Gln Ala Val Asp Ile Arg Gly Ala Asp Lys Leu Ser Ser Phe Thr Arg 465 470 475 480 Pro Leu Tyr Glu Arg Thr Arg Lys Ile Val Pro Phe Phe Asp Arg Asp 485 490 495 Glu Thr Ile Thr Asp Tyr Val Glu Lys Leu Ala Ala Asp Leu Ile Ala 500 505 510 Gly Glu Pro Val Asp Ala Ala Val Ala Ala His 515 520 31566DNAStreptomyces coelicolor 3atgttccgca gcgagtacgc agacgtcccg cccgtcgacc tgcccatcca cgacgccgtg 60ctcggcgggg ccgccgcctt cgggagcacc ccggcgctga tcgacggcac cgacggcacc 120accctcacct acgagcaggt ggaccggttc caccggcgcg tcgccgccgc cctcgccgag 180accggcgtgc gcaagggcga cgtcctcgcc ctgcacagcc ccaacaccgt cgccttcccc 240ctggccttct acgccgccac ccgcgcgggc gcctccgtca ccacggtgca tccgctcgcg 300acggcggagg agttcgccaa gcagctgaag gacagcgcgg cccgctggat cgtcaccgtc 360tcaccgctcc tgtccaccgc ccgccgggcc gccgaactcg cgggcggcgt ccaggagatc 420ctggtctgcg acagcgcgcc cggtcaccgc tccctcgtcg acatgctggc ctcgaccgcg 480cccgaaccgt ccgtcgccat cgacccggcc gaggacgtcg ccgccctgcc gtactcctcg 540ggcaccaccg gcacccccaa gggcgtcatg ctcacacacc ggcagatcgc caccaacctc 600gcccagctcg aaccgtcgat gccgtccgcg cccggcgacc gcgtcctcgc cgtgctgccg 660ttcttccaca tctacggcct gaccgccctg atgaacgccc cgctccggct cggcgccacc 720gtcgtggtcc tgccccgctt cgacctggag cagttcctcg ccgccatcca gaaccaccgc 780atcaccagcc tgtacgtcgc cccgccgatc gtcctggccc tcgccaaaca ccccctggtc 840gccgactacg acctctcctc gctgaggtac atcgtcagcg ccgccgcccc gctcgacgcg 900cgtctcgccg ccgcctgctc gcagcggctc ggcctgccgc ccgtcggcca ggcctacggc 960atgaccgaac tgtccccggg cacccacgtc gtccccctgg acgcgatggc cgacgcgccg 1020cccggcaccg tcggcaggct catcgcgggc accgagatgc gcatcgtctc cctcaccgac 1080ccgggcacgg acctccccgc cggagagtcc ggggagatcc tcatccgcgg cccccagatc 1140atgaagggct acctgggccg ccccgacgcc accgccgcca tgatcgacga ggagggctgg 1200ctgcacaccg gggacgtcgg acacgtcgac gccgacggct ggctgttcgt cgtcgaccgc 1260gtcaaggaac tgatcaagta caagggcttc caggtggccc ccgccgaact ggaggcccac 1320ctgctcaccc accccggcgt cgccgacgcg gccgtcgtcg gcgcctacga cgacgacggc 1380aacgaggtac cgcacgcctt cgtcgtccgc cagccggccg cacccggcct cgcggagagc 1440gagatcatga tgtacgtcgc cgaacgcgtc gccccctaca aacgcgtccg ccgggtcacc 1500ttcgtcgacg ccgtcccccg cgccgcctcc ggcaagatcc tccgccgaca gctcagggag 1560ccgcga 15664522PRTStreptomyces coelicolor 4Met Phe Arg Ser Glu Tyr Ala Asp Val Pro Pro Val Asp Leu Pro Ile 1 5 10 15 His Asp Ala Val Leu Gly Gly Ala Ala Ala Phe Gly Ser Thr Pro Ala 20 25 30 Leu Ile Asp Gly Thr Asp Gly Thr Thr Leu Thr Tyr Glu Gln Val Asp 35 40 45 Arg Phe His Arg Arg Val Ala Ala Ala Leu Ala Glu Thr Gly Val Arg 50 55 60 Lys Gly Asp Val Leu Ala Leu His Ser Pro Asn Thr Val Ala Phe Pro 65 70 75 80 Leu Ala Phe Tyr Ala Ala Thr Arg Ala Gly Ala Ser Val Thr Thr Val 85 90 95 His Pro Leu Ala Thr Ala Glu Glu Phe Ala Lys Gln Leu Lys Asp Ser 100 105 110 Ala Ala Arg Trp Ile Val Thr Val Ser Pro Leu Leu Ser Thr Ala Arg 115 120 125 Arg Ala Ala Glu Leu Ala Gly Gly Val Gln Glu Ile Leu Val Cys Asp 130 135 140 Ser Ala Pro Gly His Arg Ser Leu Val Asp Met Leu Ala Ser Thr Ala 145 150 155 160 Pro Glu Pro Ser Val Ala Ile Asp Pro Ala Glu Asp Val Ala Ala Leu 165 170 175 Pro Tyr Ser Ser Gly Thr Thr Gly Thr Pro Lys Gly Val Met Leu Thr 180 185 190 His Arg Gln Ile Ala Thr Asn Leu Ala Gln Leu Glu Pro Ser Met Pro 195 200 205 Ser Ala Pro Gly Asp Arg Val Leu Ala Val Leu Pro Phe Phe His Ile 210 215 220 Tyr Gly Leu Thr Ala Leu Met Asn Ala Pro Leu Arg Leu Gly Ala Thr 225 230 235 240 Val Val Val Leu Pro Arg Phe Asp Leu Glu Gln Phe Leu Ala Ala Ile 245 250 255 Gln Asn His Arg Ile Thr Ser Leu Tyr Val Ala Pro Pro Ile Val Leu 260 265 270 Ala Leu Ala Lys His Pro Leu Val Ala Asp Tyr Asp Leu Ser Ser Leu 275 280 285 Arg Tyr Ile Val Ser Ala Ala Ala Pro Leu Asp Ala Arg Leu Ala Ala 290 295 300 Ala Cys Ser Gln Arg Leu Gly Leu Pro Pro Val Gly Gln Ala Tyr Gly 305 310 315 320 Met Thr Glu Leu Ser Pro Gly Thr His Val Val Pro Leu Asp Ala Met 325 330 335 Ala Asp Ala Pro Pro Gly Thr Val Gly Arg Leu Ile Ala Gly Thr Glu 340 345 350 Met Arg Ile Val Ser Leu Thr Asp Pro Gly Thr Asp Leu Pro Ala Gly 355 360 365 Glu Ser Gly Glu Ile Leu Ile Arg Gly Pro Gln Ile Met Lys Gly Tyr 370 375 380 Leu Gly Arg Pro Asp Ala Thr Ala Ala Met Ile Asp Glu Glu Gly Trp 385 390 395 400 Leu His Thr Gly Asp Val Gly His Val Asp Ala Asp Gly Trp Leu Phe 405 410 415 Val Val Asp Arg Val Lys Glu Leu Ile Lys Tyr Lys Gly Phe Gln Val 420 425 430 Ala Pro Ala Glu Leu Glu Ala His Leu Leu Thr His Pro Gly Val Ala 435 440 445 Asp Ala Ala Val Val Gly Ala Tyr Asp Asp Asp Gly Asn Glu Val Pro 450 455 460 His Ala Phe Val Val Arg Gln Pro Ala Ala Pro Gly Leu Ala Glu Ser 465 470 475 480 Glu Ile Met Met Tyr Val Ala Glu Arg Val Ala Pro Tyr Lys Arg Val 485 490 495 Arg Arg Val Thr Phe Val Asp Ala Val Pro Arg Ala Ala Ser Gly Lys 500 505 510 Ile Leu Arg Arg Gln Leu Arg Glu Pro Arg 515 520 51032DNAArabidopsis thaliana 5atgccagtcg acgtagcctc accggccgga aaaaccgtct gcgtcaccgg agctggtgga 60tacatcgctt cttggattgt taagatactt ctcgagagag gttacacagt caaaggaacc 120gtacggaatc cagatgatcc gaagaacaca catttgagag aactagaagg aggaaaggag 180agactgattc tgtgcaaagc agatcttcag gactacgagg ctcttaaggc ggcgattgat 240ggttgcgacg gcgtctttca cacggcttct cctgtcaccg acgatccgga acaaatggtg 300gagccggccg tgaatggagc caagtttgta attaatgctg cggctgaggc caaggtcaag 360cgcgtggtca tcacctcctc cattggtgcc gtctacatgg acccgaaccg tgaccctgag 420gctgtcgttg acgaaagttg ttggagtgat cttgacttct gcaaaaacac caagaattgg 480tattgttacg gcaagatggt ggcggaacaa gcggcgtggg agacagcaaa ggagaaaggt 540gttgacttgg tggtgttgaa tccggtgctg gttcttggac cgccgttaca gccgacgatc 600aacgccagtc tttaccacgt cctcaaatat ctaaccggct cggctaagac ttatgctaat 660ttgactcaag cttatgtgga tgttcgcgat gtcgcgctgg ctcatgttct ggtctatgag 720gcaccctcgg cctccggacg ttatctccta gccgagagtg ctcgccaccg cggggaagtt 780gttgagattc tggctaagct attcccggag tatcctcttc cgaccaagtg caaggacgag 840aagaacccta gagccaagcc atacaaattc actaaccaga agattaagga cttaggctta 900gagttcactt ccaccaagca aagcctctac gacacagtca agagcttaca agagaaaggc 960catcttgctc ctcctcctcc tcctccttca gcatcgcaag aatccgtgga aaatggcatt 1020aagatcgggt ct 10326344PRTArabidopsis thaliana 6Met Pro Val Asp Val Ala Ser Pro Ala Gly Lys Thr Val Cys Val Thr 1 5 10 15 Gly Ala Gly Gly Tyr Ile Ala Ser Trp Ile Val Lys Ile Leu Leu Glu 20 25 30 Arg Gly Tyr Thr Val Lys Gly Thr Val Arg Asn Pro Asp Asp Pro Lys 35 40 45 Asn Thr His Leu Arg Glu Leu Glu Gly Gly Lys Glu Arg Leu Ile Leu 50 55 60 Cys Lys Ala Asp Leu Gln Asp Tyr Glu Ala Leu Lys Ala Ala Ile Asp 65 70 75 80 Gly Cys Asp Gly Val Phe His Thr Ala Ser Pro Val Thr Asp Asp Pro 85 90 95 Glu Gln Met Val Glu Pro Ala Val Asn Gly Ala Lys Phe Val Ile Asn 100 105 110 Ala Ala Ala Glu Ala Lys Val Lys Arg Val Val Ile Thr Ser Ser Ile 115 120 125 Gly Ala Val Tyr Met Asp Pro Asn Arg Asp Pro Glu Ala Val Val Asp 130 135 140 Glu Ser Cys Trp Ser Asp Leu Asp Phe Cys Lys Asn Thr Lys Asn Trp 145 150 155 160 Tyr Cys Tyr Gly Lys Met Val Ala Glu Gln Ala Ala Trp Glu Thr Ala 165 170 175 Lys Glu Lys Gly Val Asp Leu Val Val Leu Asn Pro Val Leu Val Leu 180 185 190 Gly Pro Pro Leu Gln Pro Thr Ile Asn Ala Ser Leu Tyr His Val Leu 195 200 205 Lys Tyr Leu Thr Gly Ser Ala Lys Thr Tyr Ala Asn Leu Thr Gln Ala 210 215 220 Tyr Val Asp Val Arg Asp Val Ala Leu Ala His Val Leu Val Tyr Glu 225 230 235 240 Ala Pro Ser Ala Ser Gly Arg Tyr Leu Leu Ala Glu Ser Ala Arg His 245 250 255 Arg Gly Glu Val Val Glu Ile Leu Ala Lys Leu Phe Pro Glu Tyr Pro 260 265 270 Leu Pro Thr Lys Cys Lys Asp Glu Lys Asn Pro Arg Ala Lys Pro Tyr 275 280 285 Lys Phe Thr Asn Gln Lys Ile Lys Asp Leu Gly Leu Glu Phe Thr Ser 290 295 300 Thr Lys Gln Ser Leu Tyr Asp Thr Val Lys Ser Leu Gln Glu Lys Gly 305 310 315 320 His Leu Ala Pro Pro Pro Pro Pro Pro Ser Ala Ser Gln Glu Ser Val 325 330 335 Glu Asn Gly Ile Lys Ile Gly Ser 340 778DNAArtificial SequenceSynthetic primer 7gcatctagaa acacaaacaa ggaaggaaga taaatgcacc accaccacca ccaccaccac 60atgccagtcg acgtagcc 78842DNAArtificial sequenceSynthetic primer 8atgcgcggcc gcttatcaag acccgatctt aatgccattt tc 42980DNAArtificial sequenceSynthetic primer 9gcatctagac cgaaatcaaa aggaacacca acgtatgcac caccaccacc accaccacca 60catggcgcca caagaacaag 801041DNAArtificial SequenceSynthetic primer 10atgcgcggcc gcttatcaca atccatttgc tagttttgcc c 411141DNAArtificial SequenceSynthetic primer 11atgcgcggcc gcttatcaca atccatttgc tagttttgcc c 411235DNAArtificial SequenceSynthetic primer 12atgcgcggcc gcttatcatc gcggctccct gagct 351380DNAArtificial SequenceSynthetic primer 13gcatctagac accttaagga ggtctatctt tcatatgcac caccaccacc accaccacca 60catggagatt aacggggcac 801439DNAArtificial SequenceSynthetic primer 14atgcgcggcc gcttatcaac atattggaat gggagctcc 391596DNAArtificial SequenceSynthetic primer 15gcattctaga cccaacgaag ggggaaccac acaatatgca ccaccaccac caccaccacc 60acaccttcgt tattgaactg gatatgaatg ttaccc 961635DNAArtificial SequenceSynthetic primer 16atgcgcggcc gcttatcagt gtgctgccac ggctg 35172175DNAArabidopsis thaliana 17atggagatta acggggcaca caagagcaac ggaggaggag tggacgctat gttatgcggc 60ggagacatca agacaaagaa catggtgatc aacgcggagg atcctctcaa ctggggagct 120gcagcggagc aaatgaaagg tagccatttg gatgaagtga agagaatggt tgctgagttt 180aggaagccag ttgtgaatct tggtggtgag actctgacca ttggacaagt

ggctgcgatc 240tcaactattg gtaacagtgt gaaggtggag ctatcggaga cagctagagc cggtgtgaat 300gctagtagtg attgggttat ggagagtatg aacaaaggca ctgatagtta tggtgttact 360actggttttg gtgctacttc tcatcggaga accaaaaacg gtgtcgcact tcagaaggaa 420cttattagat tccttaacgc cggaatattc ggaagcacga aagaaacaag ccacacattg 480ccacactccg ccacaagagc cgccatgctt gtacgaatca acactctcct ccaaggattt 540tccggtatcc gatttgagat tctcgaagca attaccagtt tcctcaacaa caacatcact 600ccatctctcc ccctccgtgg tacaatcacc gcctccggag atctcgttcc tctctcctac 660atcgccggac ttctcaccgg tcgtcccaat tccaaagcta ctggtcccaa cggtgaagct 720ttaacagcag aggaagcttt caaattagca ggaatcagct ccggattctt tgatctccag 780cctaaggaag gtctcgcgct agtcaatggc acggcggttg gatctggaat ggcgtcaatg 840gtgttattcg aaacgaatgt tctctctgtt ttggctgaga ttttgtcggc ggttttcgca 900gaggtgatga gtggtaagcc tgagttcacc gatcatctca ctcacagact taaacatcat 960cccggtcaaa tcgaagcggc ggcgataatg gagcatatcc tcgacggaag ctcgtacatg 1020aaattagctc agaagcttca cgagatggat ccgttacaga aacctaaaca agatcgttac 1080gctcttcgta cttctcctca atggttaggt cctcaaatcg aagtgatccg ttacgcaacg 1140aaatcgatcg agcgtgagat taactccgtc aacgataatc cgttgatcga tgtttcgagg 1200aacaaggcga ttcacggtgg taacttccaa ggaacaccaa tcggagtttc aatggataac 1260acgagattgg cgatagcagc gattggtaaa ctcatgtttg ctcaattctc agagcttgtg 1320aatgatttct acaacaatgg tttaccctcg aatctaaccg cttcgaggaa tccaagtttg 1380gattatggat tcaagggagc tgagattgca atggcttctt attgttcaga gcttcaatac 1440ttagctaatc ctgtgactag ccatgttcaa tcagcagagc aacataacca agatgtcaac 1500tctttgggac taatctcgtc tcgcaaaact tctgaagctg ttgatattct caagcttatg 1560tcaacaacgt tcctcgttgc gatttgtcaa gctgtggatt tgagacattt ggaggagaat 1620ttgagacaga ctgtgaagaa cactgtctct caagtggcga agaaagttct tactactgga 1680gtcaatggtg agcttcatcc ttctcgcttc tgcgaaaagg atttactcaa agttgtagac 1740cgtgaacaag tctacacata cgcggatgat ccttgtagcg caacgtaccc gttgattcag 1800aagctgagac aagttattgt tgaccatgct ttgatcaatg gtgagagtga gaagaatgca 1860gtgacttcaa tcttccataa gattggagct ttcgaggagg agcttaaggc agtgctaccg 1920aaagaagtgg aagcagcaag agcagcctac gataacggaa catcggctat cccgaacagg 1980atcaaggaat gtaggtcgta tccattgtat agattcgtga gggaagagct tggaacagag 2040cttttgaccg gagagaaagt gacgtcgcct ggagaagagt tcgacaaggt tttcacggcg 2100atttgtgaag gtaaaatcat tgatccgatg atggaatgtc tcaacgagtg gaacggagct 2160cccattccaa tatgt 2175181683DNAArabidopsis thaliana 18atggcgccac aagaacaagc agtttctcag gtgatggaga aacagagcaa caacaacaac 60agtgacgtca ttttccgatc aaagttaccg gatatttaca tcccgaacca cctatctctc 120cacgactaca tcttccaaaa catctccgag ttcgccacta agccttgcct aatcaacgga 180ccaaccggcc acgtgtacac ttactccgac gtccacgtca tctcccgcca aatcgccgcc 240aattttcaca aactcggcgt taaccaaaac gacgtcgtca tgctcctcct cccaaactgt 300cccgagttcg tcctctcttt cctcgccgcc tccttccgcg gcgcaaccgc caccgccgca 360aaccctttct tcactccggc ggagatagct aaacaagcca aagcctccaa caccaaactc 420ataatcaccg aagctcgtta cgtcgacaaa atcaaaccac ttcaaaacga cgacggagta 480gtcatcgtct gcatcgacga caacgaatcc gtgccaatcc ctgaaggctg cctccgcttc 540accgagttga ctcagtcgac aaccgaggca tcagaagtca tcgactcggt ggagatttca 600ccggacgacg tggtggcact accttactcc tctggcacga cgggattacc aaaaggagtg 660atgctgactc acaagggtct agtcacgagc gttgctcagc aagtcgacgg cgagaacccg 720aatctttatt tccacagcga tgacgtcata ctctgtgttt tgcccatgtt tcatatctac 780gctttgaact cgatcatgtt gtgtggtctt agagttggtg cggcgattct gataatgccg 840aagtttgaga tcaatctgct attggagctg atccagaggt gtaaagtgac ggtggctccg 900atggttccgc cgattgtgtt ggccattgcg aagtcttcgg agacggagaa gtatgatttg 960agctcgataa gagtggtgaa atctggtgct gctcctcttg gtaaagaact tgaagatgcc 1020gttaatgcca agtttcctaa tgccaaactc ggtcagggat acggaatgac ggaagcaggt 1080ccagtgctag caatgtcgtt aggttttgca aaggaacctt ttccggttaa gtcaggagct 1140tgtggtactg ttgtaagaaa tgctgagatg aaaatagttg atccagacac cggagattct 1200ctttcgagga atcaacccgg tgagatttgt attcgtggtc accagatcat gaaaggttac 1260ctcaacaatc cggcagctac agcagagacc attgataaag acggttggct tcatactgga 1320gatattggat tgatcgatga cgatgacgag cttttcatcg ttgatcgatt gaaagaactt 1380atcaagtata aaggttttca ggtagctccg gctgagctag aggctttgct catcggtcat 1440cctgacatta ctgatgttgc tgttgtcgca atgaaagaag aagcagctgg tgaagttcct 1500gttgcatttg tggtgaaatc gaaggattcg gagttatcag aagatgatgt gaagcaattc 1560gtgtcgaaac aggttgtgtt ttacaagaga atcaacaaag tgttcttcac tgaatccatt 1620cctaaagctc catcagggaa gatattgagg aaagatctga gggcaaaact agcaaatgga 1680ttg 1683191569DNAArtificial SequenceSynthetic phenylalanine ammonia lyase 19atgaccttcg ttattgaact ggatatgaat gttaccctgg accaactgga agatgcggcc 60cgtcagcgta ccccggtgga actgtctgcc ccggtgcgtt cccgcgtgcg tgcctcacgt 120gatgttctgg tcaaatttgt tcaggacgaa cgcgtgatct atggcgttaa cacctcgatg 180ggcggtttcg tggatcatct ggtgccggtt tcacaagcgc gtcagctgca agaaaacctg 240attaatgcgg cggcaacgaa tgtgggtgcc tacctggatg acaccacggc acgcaccatt 300atgctgtcgc gtatcgttag cctggcgcgc ggcaacagcg ctatcacgcc ggcgaatctg 360gataaactgg tcgccgtgct gaacgcaggt attgtgccgt gcatcccgga aaaaggctct 420ctgggcacca gcggcgacct gggtccgctg gctgcgatcg ctctggtttg tgcgggccag 480tggaaagccc gttataacgg ccagattatg ccgggtcgcc aagccctgtc cgaagcaggc 540gtggaaccga tggaactgtc atacaaagat ggtctggcgc tgattaatgg cacgagcggt 600atggtgggtc tgggcacgat ggtgctgcaa gcagcacgtc gcctggttga tcgctatctg 660caagtcagcg ctctgtctgt ggaaggcctg gcgggtatga ccaaaccgtt tgacccgcgt 720gttcatggcg tcaaaccgca ccgcggtcag cgtcaagttg cctctcgcct gtgggaaggc 780ctggctgata gtcacctggc ggtcaacgaa ctggacacgg aacagaccct ggcaggcgaa 840atgggcaccg tggctaaagc gggttcgctg gctattgaag atgcgtatag catccgttgc 900acgccgcaga ttctgggtcc ggtggttgat gttctggacc gcatcggtgc aaccctgcaa 960gatgaactga atagctctaa cgacaatccg attgtcctgc cggaagaagc ggaagtgttt 1020cataacggcc atttccacgg tcaatacgtg gcgatggcga tggatcacct gaatatggct 1080ctggcgaccg ttacgaacct ggctaatcgt cgcgtcgatc gttttctgga caaatcaaac 1140tcgaatggtc tgccggcctt cctgtgtcgt gaagatccgg gtctgcgtct gggtctgatg 1200ggcggtcaat ttatgacggc ctctatcacc gcagaaaccc gtacgctgac cattccgatg 1260agtgtgcagt ccctgacgtc aaccgcggat ttccaagaca tcgttagttt tggtttcgtc 1320gctgcacgtc gcgcccgcga agtcctgacc aatgccgcat acgtcgtggc atttgaactg 1380ctgtgcgcct gtcaggcagt tgatattcgt ggtgcggaca aactgagttc cttcacgcgc 1440ccgctgtatg aacgcacccg taaaatcgtc ccgtttttcg atcgcgacga aacgattacc 1500gattacgtgg aaaaactggc agccgacctg attgcgggtg aaccggtgga tgcagccgtg 1560gcagcacac 1569

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